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nAture methods | VOL.12 NO.12 | DECEMBER 2015 | 1143
epigenome editing with the crisPr (clustered, regularly interspaced, short palindromic repeats)-cas9 platform is a promising technology for modulating gene expression to direct cell phenotype and to dissect the causal epigenetic mechanisms of gene regulation. Fusions of nuclease-inactive dcas9 to the Krüppel-associated box (KrAB) repressor (dcas9-KrAB) can silence target gene expression, but the genome-wide specificity and the extent of heterochromatin formation catalyzed by dcas9-KrAB are not known. We targeted dcas9-KrAB to the hs2 enhancer, a distal regulatory element that orchestrates the expression of multiple globin genes, and observed highly specific induction of h3K9 trimethylation (h3K9me3) at the enhancer and decreased chromatin accessibility of both the enhancer and its promoter targets. targeted epigenetic modification of hs2 silenced the expression of multiple globin genes, with minimal off-target changes in global gene expression. these results demonstrate that repression mediated by dcas9-KrAB is sufficiently specific to disrupt the activity of individual enhancers via local modification of the epigenome.
Custom control over epigenetic regulation is becoming increas-ingly attainable with the expansion of genome engineering tech-nologies that combine epigenetic modulators with programmable DNA-targeting platforms. Engineered zinc-finger proteins, tran-scription activator–like effectors and the CRISPR-dCas9 system can localize effector domains to target genomic regions in order to control epigenetic states1–12. In the CRISPR-dCas9 system adapted from Streptococcus pyogenes, codelivery of a single guide RNA (sgRNA) directs dCas9 binding through an 18–20-nt protospacer region complementary to the target genomic sequence13–15. The ease and versatility of this RNA-guided epigenome-editing plat-form have enabled its rapid application for designing gene regula-tory networks, screening for cellular phenotypes and directing cell fate9,16–21. Site-specific epigenome engineering with the CRISPR-dCas9 system is also a promising technology for investigating the function of distal regulatory elements9,10. Large-scale efforts to map the human epigenome have revealed more than 1 million
DNase I–hypersensitive sites (DHSs), many of which probably act as cell-type-specific enhancers22–24. Epigenome editing pro-teins can be targeted to candidate regulatory elements in order to modify local chromatin structure and determine the role of these distal elements in influencing endogenous gene expression.
For targeted gene repression, the most commonly used effector is the KRAB domain1,3,6. When localized to DNA, KRAB recruits a heterochromatin-forming complex that causes histone meth-ylation and deacetylation25–28. dCas9-KRAB fusions have effec-tively silenced noncoding RNAs and single genes when targeted to promoter regions, 5′ untranslated regions and proximal enhancer elements6,9,16,18. Libraries of sgRNAs targeting dCas9-KRAB to genomic regions proximal to transcription start sites have also been used for high-throughput gene-silencing screens16.
Unmodified dCas9 is known to bind at off-target loci contain-ing a 5-bp seed sequence followed by a protospacer-adjacent motif29,30. The potential for binding and subsequent epigenome editing by dCas9-KRAB at off-target sites has not been evaluated. Off-target activity is a particular concern given recent evidence that the addition of a KRAB domain to a targeted zinc-finger protein can dramatically increase off-target interactions31. Other studies also suggest that targeting the KRAB domain to genomic loci with other DNA-binding proteins can lead to long-range, stable H3K9me3 and chromatin condensation2,25–27,32,33. If similar issues arise with the dCas9-KRAB platform, long-range epigenetic effects will convolute loss-of-function screens and the annotation of gene regulatory elements. Therefore, we sought to evaluate the genome-wide specificity of gene regulation, DNA binding and chromatin remodeling catalyzed by dCas9-KRAB targeted to an endogenous distal regulatory element.
We targeted dCas9-KRAB to the HS2 enhancer in the globin locus control region (LCR)34. The LCR contains five DHS enhancer regions (HS1–5) that orchestrate the expression of hemoglobin subunit genes in erythroid cells during development. Studies of HS2 have revealed fundamental mechanisms of enhancer activity such as transcription-factor binding, maintenance of chromatin accessibility, and chromatin looping to globin
1Department of Biomedical Engineering, Duke University, Durham, North Carolina, USA. 2Center for Genomic and Computational Biology, Duke University, Durham, North Carolina, USA. 3University Program in Genetics and Genomics, Duke University, Durham, North Carolina, USA. 4Department of Pediatrics, Division of Medical Genetics, Duke University Medical Center, Durham, North Carolina, USA. 5Department of Biostatistics & Bioinformatics, Duke University Medical Center, Durham, North Carolina, USA. 6Department of Orthopaedic Surgery, Duke University Medical Center, Durham, North Carolina, USA. Correspondence should be addressed to T.E.R. ([email protected]), G.E.C. ([email protected]) or C.A.G. ([email protected]).Received 30 ApRil; Accepted 19 SeptembeR; publiShed online 26 octobeR 2015; doi:10.1038/nmeth.3630
highly specific epigenome editing by crisPr-cas9 repressors for silencing of distal regulatory elementsPratiksha I Thakore1,2, Anthony M D’Ippolito2,3, Lingyun Song2,4, Alexias Safi2,4, Nishkala K Shivakumar1, Ami M Kabadi1,2, Timothy E Reddy2,5, Gregory E Crawford2,4 & Charles A Gersbach1,2,6
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promoters35–38. Here we demonstrate that targeting dCas9-KRAB to the HS2 enhancer disrupted the expression of multiple globin genes. Genome-wide analysis of dCas9-KRAB binding and repression activity established that RNA-guided synthetic repres-sors are highly specific for endogenous target loci. Deposition of H3K9me3 was limited to the intended HS2 region, leading to decreased chromatin accessibility at both the targeted enhancer and its associated promoters. These results demonstrate that dCas9-KRAB can alter the local epigenome of individual enhanc-ers, and they support the use of dCas9-KRAB as a highly specific epigenome editing tool for directing cell phenotype and revealing connections between regulatory elements and gene expression.
resultsdcas9-KrAB silences globin genes from a distal enhancerTo find optimal sgRNAs for repression of the HS2 enhancer by dCas9-KRAB, we designed a panel of 21 sgRNAs (Cr1–Cr21) to cover the 400-bp core of the HS2 enhancer (Fig. 1a and Supplementary Table 1). Each sgRNA was transiently transfected into human K562 erythroid leukemia cells that were modified to stably express dCas9 or dCas9-KRAB (Supplementary Fig. 1a). The activity of each sgRNA was screened 3 d after transfection by quantitative real-time PCR (qRT-PCR) for expression of HBE1, HBG1 and HBG2 (HBG1/2), and HBB mRNA (Supplementary Fig. 1b–d). Because of the sequence similarity between HBG1 and HBG2, PCR primers do not distinguish between the two transcripts. When transfected into K562 cells expressing dCas9-KRAB, most of the 21 sgRNAs caused decreased expression of HBE1 and HBG1/2. The sgRNAs had no effect on globin gene expression in unmodified K562 cells or when designed to target the IL1RN promoter as a negative control7, demonstrating that the effects were both HS2- and dCas9-specific. Reduced globin gene expression probably resulted from a combination of steric blocking and epigenome modification because dCas9-KRAB was more effective than dCas9 in silencing HBE1 and HBG1/2. HBB is not highly expressed in K562 cells39, and transient delivery of sgRNAs did not affect HBB expression. The repressive effects did not persist at 6 d after transient transfection of sgRNA expression plasmids (Supplementary Fig. 2), likely because of a time-dependent reduction of the transfected DNA.
For further characterization of the mechanisms of targeted repression by dCas9 and dCas9-KRAB, four sgRNAs
from the initial screen with the strongest effects on globin gene expression (Cr2, Cr4, Cr7 and Cr10) were transferred into a lentiviral vector and coexpressed with either dCas9 or dCas9-KRAB (Fig. 1b). Stable coexpression of each of the four sgRNAs with dCas9-KRAB substantially repressed expression of HBE1, HBG1/2 and HBB 7 d after transduction relative to codeliv-ery of the sgRNA with dCas9 (Fig. 1c–e). Globin gene expression was not altered by dCas9 or dCas9-KRAB delivered alone or with an IL1RN-targeted sgRNA. As observed with transient sgRNA delivery, targeting dCas9-KRAB to HS2 decreased globin expres-sion more than targeting dCas9 did. Moreover, stable expression of HS2-targeted sgRNAs with dCas9-KRAB, but not with dCas9, decreased HBG1/2 protein expression over 21 d (Supplementary Fig. 3). These results demonstrate that dCas9-KRAB repressors targeted by a single sgRNA can interrupt distal enhancer activity and silence the expression of multiple genes >10 kb away.
dcas9-KrAB repression is highly specificPrevious studies using multiplexed sgRNAs with dCas9-VP64 activators have established the specificity of endogenous gene activation7,40. Additionally, sgRNA-mediated silencing with dCas9-KRAB is highly specific, as determined by microarray analyses in experiments targeting an endogenous promoter9 and RNA-seq in experiments targeting a reporter gene6. To determine whether that specificity extends to dCas9-KRAB– mediated repression of an endogenous enhancer, we used RNA-seq to measure the transcriptome-wide effects of targeting dCas9-KRAB and dCas9 to HS2 using the Cr4 and Cr10 sgRNAs (Fig. 2a,b and Supplementary Fig. 4a,b).
Compared with targeting with dCas9-KRAB alone, when dCas9-KRAB was targeted to the HS2 enhancer by either Cr4 or Cr10, the only significantly repressed genes were HBG1/2, HBE1 and HBBP1 (false discovery rate (FDR) < 0.01) (Fig. 2a,b). We observed a similar result when we compared the effect of target-ing dCas9-KRAB to that of targeting dCas9, both codelivered with sgRNAs (Supplementary Fig. 4a,b). HBBP1 is a pseudo-gene that does not encode functional globin protein but may still be regulated by HS2. HBD expression was also reduced by targeting of dCas9-KRAB to HS2 with either Cr4 or Cr10, but the change in expression was not shown to be significant after
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Figure 1 | Silencing of downstream globin genes by dCas9-KRAB transcription factors targeted to the distal HS2 enhancer. (a,b) A panel of 21 sgRNAs was designed to target dCas9-KRAB to the HS2 enhancer (a). Cr2, Cr4, Cr7 and Cr10 sgRNAs were selected for further study and stably delivered to K562 cells using the lentiviral vector shown in b. (c–e) Repression of the HS2 enhancer was assayed by qRT-PCR of (c) HBE1, (d) HBG1/2 and (e) HBB, and fold-changes were calculated relative to nontransduced K562 cells (mean ± s.e.m.). In c–e, groups that share the same letter (A–F) are not significantly different as determined by multiway analysis of variance followed by Tukey’s post hoc test; P < 0.05 (n = 3 independent experiments).
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multiple-hypothesis testing. The only significant off-target effect observed was an increase in expression of PCSK1N when dCas9-KRAB + Cr10 was compared to dCas9 + Cr10 (P = 1.6 × 10−7 Supplementary Fig. 4b). PCSK1N regulates processing of neuro-endocrine pathway proteins and is not known to be involved in KRAB-mediated effects. No off-target gene expression differences were observed after treatment with Cr4.
When we compared samples treated with sgRNA and either dCas9 or dCas9-KRAB to untransduced controls, we detected a greater number of differentially expressed genes, varying from six to ten depending on the comparison (Fig. 2c,d, Supplementary Fig. 4c,d and Supplementary Tables 2–5). When we compared dCas9-KRAB without guide RNA to untransduced controls, we observed 29 differentially expressed genes (Supplementary Fig. 4e and Supplementary Table 6). Three genes were differen-tially expressed relative to untreated controls in all comparisons, with or without the presence of guide RNA or the KRAB domain. These modest off-target changes in gene expression might have been the result of lentiviral transduction, puromycin selection or dCas9 expression. Overall, these results indicate that RNA-guided KRAB-mediated gene repression is highly specific, and they emphasize that the inclusion of proper controls such as dCas9-KRAB treatment without a functional sgRNA are critical to proper experimental design and interpretation.
dcas9-KrAB binding is highly specificPrevious reports have shown evidence that dCas9 promiscu-ously binds the genome outside of the target site29,30,41. However, recent studies have demonstrated that dCas9 and dCas9-KRAB
effectors can bind specifically when targeting endogenous gene promoters41. Evaluating off-target localization and poten-tial downstream effects of targeting endogenous enhancers is critical for implementing dCas9-KRAB to control cell phenotype and to investigate epigenome function. To evaluate the genome-wide DNA-binding of dCas9-KRAB, we performed ChIP-seq using a Flag epitope expressed on the N terminus of dCas9 or dCas9-KRAB (Fig. 1b). Recruitment of dCas9 and dCas9-KRAB by Cr4 or Cr10 was highly localized to the HS2 enhancer (Fig. 3a), and there were no additional significant dCas9-KRAB binding sites in the human genome other than the sgRNA target site (Fig. 3b,c and Supplementary Tables 7 and 8).
The addition of KRAB to dCas9 did not alter the strength of the binding signal (Supplementary Fig. 5a,b). With a genome-wide FDR < 0.05, there was one genomic window, located on chromo-some 2, with a significant decrease in ChIP-seq signal for dCas9-KRAB + Cr4 versus dCas9 + Cr4 (Supplementary Table 9). The
region contained a 5-bp protospacer seed sequence matched to Cr4 (refs. 29,30). No genome-wide significant changes in ChIP-seq signal were observed in comparisons of dCas9-KRAB + Cr10 versus dCas9 + Cr10 (Supplementary Fig. 5a,b). When we compared dCas9-KRAB + Cr4 or Cr10
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Figure 2 | Specificity of gene regulation by dCas9-KRAB repressors targeted to the HS2 enhancer. RNA-seq was performed for genome-wide analysis of HS2 sgRNA silencing specificity. (a–d) Differential-expression analyses of globin genes comparing dCas9-KRAB targeted by (a) Cr4 and (b) Cr10 versus dCas9-KRAB without sgRNA and comparing dCas9-KRAB guided by (c) Cr4 and (d) Cr10 to no-lentivirus control (no LV CTL) in K562 cells. Red data points indicate FDR < 0.01 by differential-expression analysis compared with dCas9-KRAB controls without sgRNA (n = 3 biological replicates). Blue data points indicate other globin genes.
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Figure 3 | Genome-wide binding activity of dCas9 repressors targeted to the HS2 enhancer. (a) ChIP-seq tracks for binding of Flag-tagged dCas9 and dCas9-KRAB to the HS2 enhancer (shaded region) of the globin locus (chr11:5,244,651–5,314,450), compared with binding of dCas9-KRAB without sgRNA. An ENCODE K562 DNase I–hypersensitivity DNase-seq track is included to highlight the globin LCR22. (b,c) Differential analyses of global binding activity including comparisons of dCas9-KRAB targeted by (b) Cr4 and (c) Cr10 versus dCas9-KRAB without sgRNA. Red data points indicate FDR < 0.05 by differential DESeq analysis (n = 3 biological replicates).
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to dCas9 + Cr4 or Cr10, we observed a decrease in ChIP-seq signal at HS2, but it did not reach genome-wide significance (P = 4.5 × 10−4 and P = 1.9 × 10−4 for Cr4 and Cr10, respectively) (Supplementary Fig. 5a,b). The decrease in Flag ChIP-seq signal at the HS2 enhancer and off-target site was perhaps due to epitope or target-site inaccessibility after formation of the repressive complex by KRAB.
dcas9-KrAB binding disrupts transcription-factor bindingWe hypothesized that targeting dCas9-KRAB to HS2 would decrease binding of endogenous transcription factors at the enhancer. The GATA2 and AP-1 transcription factors are well known to bind the HS2 enhancer and regulate globin gene expres-sion, as confirmed in K562 cells by data from the ENCODE project24,42. The binding motifs for both GATA2 and AP-1 are adjacent to the Cr4 and Cr10 sgRNA targets in the enhancer34,35,42 (Supplementary Fig. 6a). We used ChIP-qPCR to evaluate dis-ruption of the interactions of GATA2 and the AP-1 subunit FOSL1 with HS2 by dCas9-KRAB or dCas9. Recruiting dCas9-KRAB to HS2 with Cr4 or Cr10 significantly decreased binding of both GATA2 (Cr4, P = 0.0090; Cr10, P = 0.034; Supplementary Fig. 6b) and FOSL1 (Cr4, P = 0.0090; Cr10, P = 0.0087; Supplementary Fig. 6c) compared with that in dCas9-KRAB–only controls. In contrast, localizing dCas9 to the HS2 enhancer did not signifi-cantly decrease the binding of endogenous transcription factors.
dcas9-KrAB induces histone methylation at enhancersTo investigate the epigenetic consequences of KRAB-mediated silencing, we assayed global H3K9me3 patterns by ChIP-seq (Fig. 4). dCas9-KRAB targeted to endogenous promoters can induce repressive histone methylation9. Furthermore, SETDB1, a meth-yltransferase recruited by the KRAB repression complex, cata-lyzes H3K9me3 when localized to genomic loci27. Codelivery of dCas9-KRAB and either Cr4 or Cr10 significantly increased the H3K9me3 signal at the target HS2 enhancer relative to delivery of dCas9-KRAB without an sgRNA (Cr4, P = 2.01 × 10−26; Cr10, P = 1.15 × 10−23; Fig. 4b,c, Supplementary Figs. 7a,b and 8, and
Supplementary Tables 10 and 11). The strength of the H3K9me3 ChIP-seq signal induced by dCas9-KRAB at the HS2 enhancer was comparable to that of the nearby endogenous H3K9me3 signal at sites that flanked the globin locus (Fig. 4a), indicat-ing that dCas9-KRAB induced histone methylation at physio-logically relevant levels. Furthermore, the H3K9me3 observed at HS2 did not spread beyond the DHSs overlapping the enhancer, covering a span of 853 bp. The effect at HS2 was specific to the KRAB domain, as no changes in H3K9me3 occurred at the enhancer when dCas9 was targeted with Cr4 or Cr10 (Fig. 4d, Supplementary Fig. 7c–f and Supplementary Tables 12 and 13). For Cr4, an increased H3K9me3 signal was also observed at the immediately adjacent HS3 enhancer, and both Cr4 and Cr10 increased H3K9me3 at the DHSs between HS1 and HS2. Together, these results demonstrate that dCas9-KRAB can induce histone modifications associated with heterochromatin when targeted by sgRNAs to active enhancers.
To identify off-target H3K9me3 sites, we first compared dCas9-KRAB with either Cr4 or Cr10 to dCas9-KRAB without an sgRNA. For Cr4, ten off-target changes in H3K9me3 signal were detected, and eight of them contained a protospacer seed-sequence match for Cr4 (Fig. 4b and Supplementary Table 10). Only one off-target change in H3K9me3, found in IRF3, coincided with dCas9-KRAB binding by anti-Flag ChIP-seq with an FDR < 0.10 (FDR = 0.0785; Fig. 3b and Supplementary Table 7). For Cr10, one off-target H3K9me3 site was observed near CYP17A1, and this region did not contain a match for the protospacer target seed (Fig. 4c and Supplementary Table 11). Although we expected KRAB to cause only increases in H3K9me3, 5 of
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Figure 4 | Genome-wide H3K9me3 signal in K562 cells treated with dCas9-KRAB targeted to the HS2 enhancer. (a) ChIP-seq tracks for H3K9me3 signal at the HS2 enhancer (shaded area) and flanking DHS sites in the globin LCR (chr11:5,241,410–5,317,466). An ENCODE K562 DNase I–hypersensitivity DNase-seq track is included to highlight the globin LCR22. (b,c) Global analysis of H3K9me3 patterns was performed by ChIP-seq for (b) Cr4 and (c) Cr10, comparing dCas9-KRAB with sgRNA versus dCas9-KRAB without sgRNA. Red data points indicate FDR < 0.05 by differential-expression analysis compared to dCas9-KRAB without sgRNA (n = 3 biological replicates). (d) Read counts for the HS2 enhancer (chr11:5,301,862–5,302,715) from MACS-based peak calls normalized to total counts (mean ± s.e.m.). *P < 0.05 by Student’s t-test compared with dCas9-KRAB–only control.
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the 11 total off-target regions (45%) had decreased H3K9me3 signals. All changes in H3K9me3 signal outside the globin LCR were smaller in magnitude and less statis-tically significant than those at sites in the LCR (Supplementary Fig. 7a,b).
We performed a similar analysis to iden-tify differences in H3K9me3 when KRAB is added to dCas9. For the Cr4 guide RNA, we identified 44 off-target changes in H3K9me3 (Supplementary Fig. 7c). Of these changes, 39 (89%) had a decrease in ChIP-seq H3K9me3 signal that we do not think was directly due to KRAB-domain activity. The off-target changes were also low in magnitude compared with changes in the LCR (Supplementary Fig. 7c,e). We further analyzed the 20 off-target regions with the strong-est statistical significance and found that 14 contained Cr4 seed sequences (Supplementary Table 12). When we compared dCas9-KRAB + Cr10 with dCas9 + Cr10, we did not identify any off-target H3K9me3 modifications (Supplementary Fig. 7d,f and Supplementary Table 13). Overall, these results show that dCas9-KRAB generated H3K9me3 at the globin LCR with nearly perfect specificity, and off-target histone-modification events were typically subtle and varied depending on sgRNA target sequence. As many of the off-target H3K9me3 changes were decreases in H3K9me3 signal, it might be that these were false positives at this threshold of statistical significance. Furthermore, we did not observe any down-stream effects on nearby gene expression associated with off-target H3K9me3 (Fig. 2a,b and Supplementary Fig. 4a,b).
dcas9-KrAB decreases chromatin accessibility at enhancersAs an orthogonal measure of the specificity of epigenome editing by dCas9-KRAB, we used DNase I–hypersensitive sequencing
(DNase-seq) to identify genome-wide changes in chromatin accessibility concurrent with KRAB-mediated gene silencing. Targeting dCas9-KRAB to the HS2 enhancer decreased chroma-tin accessibility at HS2, HS3 and the DHSs between HS1 and HS2 (Fig. 5 and Supplementary Fig. 9a–d), which were the same regions with increases in H3K9me3 (Fig. 4a). We also observed decreased chromatin accessibility at the HBE1, HBG1 and HBG2 promoters (Fig. 5b and Supplementary Fig. 9e–h). These pro-moters are up to 25 kb from the targeted enhancer region, indicat-ing that dCas9-KRAB–mediated H3K9me3 of the LCR directly affects chromatin structure at the target promoters.
The most significant and highest-magnitude changes for both Cr4 and Cr10 occurred at the HS2 and HS3 enhancers and the HBG1 and HBG2 promoters (Fig. 5f,g and Supplementary Tables 14 and 15). The effects on chromatin state were largely dependent on the addition of the KRAB domain to dCas9, as supported by comparisons of dCas9-KRAB to dCas9 with the same guide RNAs (Fig. 5b,c and Supplementary Figs. 9a–h and 10a–d).
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chr11:5,271,045–5,271,435 (HBG1)
chr11:5,301,930–5,202,230(Cr4, HS2)
chr11:5,301,772–5,202,032(Cr4, HS2)
chr11:5,305,942–5,306,243 (HS3)
chr11:5,275,809–5,276,109(HBG2)
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f dCas9-KRAB + Cr4 vs. dCas9-KRAB only
chr11:5,270,905–5,271,205 (HBG1)
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Figure 5 | Changes in global chromatin landscape with dCas9-KRAB localized to the HS2 distal enhancer. (a) Genome-browser tracks of DNase-seq alignments at the globin locus (chr11:5,244,651–5,314,450) comparing the indicated conditions. Red shading labels the HBG1 promoter, HBG2 promoter, HS2 enhancer and HS3 enhancer regions for dCas9-KRAB + Cr4 or Cr10. (b,c) Normalized DNase-seq cut counts within an 800-bp window surrounding the (b) HBG2 promoter and (c) HS2 enhancer (mean ± s.e.m., n = 3 biological replicates). *P < 0.05 compared with the dCas9-KRAB only sample (Student’s t-test). (d,e) Differential genome-wide analysis of changes in chromatin accessibility induced by dCas9-KRAB targeted by (d) Cr4 and (e) Cr10 compared with dCas9-KRAB without sgRNA in K562 cells. (f,g) Volcano plots of significance (P value) versus fold change for differential DESeq expression analysis of dCas9-KRAB guided by (f) Cr4 or (g) Cr10 compared with dCas9-KRAB without sgRNAs. Red data points indicate FDR < 0.05 by DESeq analysis. Blue data points indicate other regions in the globin promoters or globin LCR.
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No significant effects on chromatin accessibility were found outside the globin locus, providing further evidence that dCas9-KRAB targeting has highly specific effects on the epigenome. Collectively these findings suggest substantial and highly specific changes to chromatin accessibility concomitant with gene repres-sion and the introduction of repressive histone modifications by the targeted dCas9-KRAB.
discussionThe simplicity and efficacy of the RNA-guided CRISPR-dCas9 targeting system have the potential to transform epigenome edit-ing into a widely used research tool, but the success of this applica-tion relies on precise editing of the epigenetic state. Programmable KRAB-fusion proteins have been used to repress enhancers up to 20 kb from a single target gene9,18. In this study we expanded the capability of synthetic gene regulation by targeting dCas9-KRAB to an enhancer located 10 to 50 kb away from target genes and by comprehensively characterizing the remarkable specificity of this technology.
A recent study showed that targeting dCas9-KRAB to a puta-tive distal enhancer silenced downstream gene expression but did not induce repressive histone marks at the target enhancer9. Here we demonstrate that dCas9-KRAB can deposit H3K9me3 at endogenous enhancers, indicating that dCas9-KRAB–mediated histone methylation may be dependent on locus or cell type. We also did not find evidence that addition of the KRAB domain to dCas9 increased off-target binding, in agreement with other recent dCas9-KRAB genome-wide binding studies41. This is in contrast to another study of engineered zinc finger– KRAB fusion proteins31. The difference may be due to the dis-tinct mechanism of protein-DNA binding versus RNA-DNA interactions mediated by dCas9 or locus- or cell type–specific effects. Overall, our findings advocate for the use of dCas9-KRAB repressors to achieve precise silencing of endogenous genomic targets.
Histone methylation induced by KRAB or HP1 has been esti-mated to spread up to 20 kb from the repressor binding site in syn-thetic reporter assays with other DNA-targeting proteins26,32,33. In contrast, with dCas9-KRAB, increases in H3K9me3 were found up to 4.5 kb away from the sgRNA target site. Furthermore, our H3K9me3 ChIP-seq data show that epigenome modification occurred only at flanking DHSs and did not span the region between those sites. That observation suggests that heterochro-matin spreading induced by KRAB might not occur linearly along the genome, and instead might spread through three- dimensional interactions between discrete nearby open chromatin sites. Spreading of histone methylation over long distances may therefore be site specific and dependent on three-dimensional chromatin structure at those sites.
The DNase I–hypersensitivity profiles of enhancers correlate with associated promoters22, and chromatin-conformation anal-yses have shown that the globin LCR physically interacts with HBG1, HBG2 and HBE1 in K562 cells38. Furthermore, forcing DNA-looping interactions with the globin LCR activates develop-mentally silenced embryonic and fetal globin expression43. When dCas9-KRAB was targeted to HS2, promoter regions of HBG1 and HBG2 also had decreased DNase-seq signal, whereas the HS1 region between HS2 and the target promoters was not affected. Additionally, HBG1 and HBG2 promoters did not have increased
H3K9me3 signal. The decreased hypersensitivity observed at HBG1 and HBG2 is therefore unlikely to have been caused by het-erochromatin spreading from the HS2 enhancer; instead it might be the result of disruption of long-range interactions between the promoters and the HS2 enhancer. Potential mechanisms for the disruption of DNA looping include interference with endogenous transcription-factor binding and alteration of the chromatin state of the enhancer region. Overall, these results suggest that the acces-sible chromatin structure and the active state of some promoters require continuous input from enhancer elements, and they sup-port the use of dCas9-KRAB as a potential method to identify and disrupt these enhancer-promoter interactions.
The KRAB domain is distinctive from epigenome-editing effectors because, rather than catalyzing a single type of histone modification, it draws a diverse group of histone modifiers that cooperate to form heterochromatin25,27,28,32. This broad efficacy probably contributes to the effectiveness of KRAB at silencing different types of genomic elements, including transcribed genes and proximal and distal regulatory elements6,9,16,18. This versatil-ity, along with the exceptional specificity demonstrated in this study, distinguishes dCas9-KRAB as a promising platform for applications such as gene therapy, cellular reprogramming and high-throughput screens of regulatory-element function and modulators of cell phenotype16.
methodsMethods and any associated references are available in the online version of the paper.
Accession codes. Raw RNA-seq (GSE71557), ChIP-seq (GSE70671) and DNase-seq (GSE70973) data are available in the Gene Expression Omnibus.
Note: Any Supplementary Information and Source Data files are available in the online version of the paper.
AcKnoWledgmentsWe thank the Duke Genome Sequencing & Analysis Core for sequencing the RNA-seq, ChIP-seq and DNase-seq libraries. This work was supported by US National Institutes of Health (NIH) grants R01DA036865, U01HG007900, R21AR065956 and P30AR066527; an NIH Director’s New Innovator Award (DP2OD008586); a US National Science Foundation (NSF) Faculty Early Career Development (CAREER) Award (CBET-1151035); and an American Heart Association Scientist Development grant (10SDG3060033) to C.A.G. P.I.T. was supported by an NSF Graduate Research Fellowship and an American Heart Association Mid-Atlantic Affiliate Predoctoral Fellowship.
Author contriButionsP.I.T., G.E.C., T.E.R. and C.A.G. designed experiments. P.I.T., A.M.D., A.S., N.K.S. and A.M.K. performed the experiments. P.I.T., A.M.D., L.S., A.M.K., G.E.C., T.E.R. and C.A.G. analyzed the data. P.I.T., G.E.C., T.E.R. and C.A.G. wrote the manuscript. All authors contributed to editing of the manuscript.
comPeting FinAnciAl interestsThe authors declare competing financial interests: details are available in the online version of the paper.
reprints and permissions information is available online at http://www.nature.com/reprints/index.html.
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online methodsPlasmids. The sgRNA plasmid and lentiviral plasmid encoding dCas9 are available from Addgene44 (53188 and 53191, respec-tively). The KRAB domain was cloned in-frame with the dCas9 open reading frame at the C terminus using NheI sites. For sgRNA screening, the oligonucleotides containing HS2 proto-spacer sequences were synthesized (IDT-DNA), hybridized, phosphorylated and inserted into phU6-gRNA plasmids using BbsI sites. The protospacer target sequences for the panel of 21 HS2 enhancer sgRNAs are provided in Supplementary Table 1. U6-sgRNA expression cassettes were transferred in reverse orientation upstream of the hUbC promoter at the PacI sites. For sgRNA and dCas9 transduction experiments, a puromycin resistance cassette was linked to the dCas9 and dCas9-KRAB effectors using a T2A ribosome-skipping peptide.
Cell culture. K562 cells and HEK293T cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cancer Center Facilities. Cell lines were verified via morphological inspection. K562 cells were maintained in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin. HEK293T cells were cultured in DMEM supple-mented with 10% FBS and 1% penicillin-streptomycin. All cell lines were cultured at 37 °C with 5% CO2.
Lentiviral transduction. K562 cells were transduced with lenti-virus to stably express dCas9 or dCas9-KRAB. To produce VSV-G pseudotyped lentivirus, we plated HEK293T cells at a density of 5.1 × 103 cells/cm2 in high-glucose DMEM (Gibco, 11995) sup-plemented with 10% FBS and 1% penicillin-streptomycin. The next day after seeding, cells in 10-cm plates were cotransfected with the appropriate dCas9 or dCas9-KRAB lentiviral expres-sion plasmid (20 µg), the second-generation packaging plasmid psPAX2 (Addgene, 12260, 15 µg), and the envelope plasmid pMD2.G (Addgene, 12259, 6 µg) by calcium phosphate precipi-tation45. After 14–20 h, the transfection medium was exchanged for 10 mL of fresh 293T medium. Conditioned medium con-taining lentivirus was collected 24 and 48 h after the first media exchange. Residual producer cells were cleared from the lentiviral supernatant by filtration through 0.45-µm cellulose acetate filters and concentrated 20-fold by centrifugation through a 100-kDa molecular-weight-cutoff filter (Millipore). Concentrated viral supernatant was snap-frozen in liquid nitrogen and stored at −80 °C for future use. For transduction, concentrated viral super-natant was diluted 1:10 with K562 media. To facilitate transduc-tion, we added the cationic polymer polybrene to the viral media at a concentration of 4 µg/mL. Nontransduced (NT) cells did not receive virus but were treated with polybrene as a control. The day after transduction, the medium was exchanged to remove the virus. For cells that were transduced with lentivirus containing a puromycin-resistance gene, 1 µg/ml puromycin was used to initiate selection for transduced cells approximately 96 h after transduction.
Transient transfection. Six to eight days after transduction with dCas9 or dCas9-KRAB lentivirus, K562 cells were transiently transfected with plasmid encoding HS2 sgRNAs via electropo-ration. 2× 106 cells were transfected with 5 µg of plasmid DNA in 200 uL of Opti-MEM (Gibco) in 2-mm cuvettes at 160 V,
950 µF and infinite resistance. Transfection efficiencies of greater than 70% were routinely achieved, as assayed by flow cytometry after delivery of a control eGFP expression plasmid (unpublished data, P.I. Thakore).
Western blotting. Cells were lysed in RIPA buffer (Sigma), and a BCA assay (Pierce) was performed to quantify total pro-tein. Lysates were mixed with LDS sample buffer (Invitrogen) and boiled for 5 min; equal amounts of total protein were run in NuPAGE Novex 4–12% Bis-Tris polyacrylamide gels (Life Technologies) and transferred to nitrocellulose membranes. Nonspecific antibody binding was blocked with 5% nonfat milk in TBS-T (50 mM Tris, 150 mM NaCl and 0.1% Tween-20) for 30 min. The membranes were then incubated with primary anti-bodies in 5% milk in TBS-T: mouse anti-γ hemoglobin subu-nit (Santa Cruz, clone 51-7) diluted 1:1,000 overnight at 4 °C, anti-Flag (Sigma, F7425) diluted 1:1,000 for 60 min at room temperature, or rabbit anti-GAPDH (Cell Signaling, clone 14C10) diluted 1:5,000 for 60 min at room temperature. Membranes labeled with primary antibodies were incubated with anti-mouse (Santa Cruz, SC-2005) or anti-rabbit HRP-conjugated antibody (Sigma-Aldrich, A6154) diluted 1:5,000 for 60 min and washed with TBS-T for 60 min. Membranes were visualized using the Immun-Star WesternC Chemiluminescence Kit (Bio-Rad), and images were captured using a ChemiDoc XRS+ system and processed using ImageLab software (Bio-Rad).
Quantitative reverse-transcription PCR. Cells were harvested for total RNA isolation using the RNeasy Plus RNA isolation kit (Qiagen). cDNA synthesis was performed using the SuperScript VILO cDNA Synthesis Kit (Invitrogen). qRT-PCR using QuantIT Perfecta Supermix was performed with the CFX96 Real-Time PCR Detection System (Bio-Rad) with the oligonucleotide primers reported in Supplementary Table 16. The results are expressed as fold-increase mRNA expression of the gene of interest normalized to GAPDH expression by the ∆∆Ct method.
RNA sequencing. RNA-seq libraries were constructed as pre-viously described46. Briefly, 7 d after transduction, cells were harvested and mRNA was purified from total RNA using oligo(dT) Dynabeads (Invitrogen). First-strand cDNA was synthesized using the SuperScript VILO cDNA Synthesis Kit (Invitrogen), and second-strand cDNA was synthesized using DNA polymerase I (New England BioLabs). cDNA was purified using Agencourt AMPure XP beads (Beckman Coulter). Purified cDNA was treated with Nextera transposase (Illumina) for 5 min at 55 °C to simultaneously fragment and insert sequencing primers into the double-stranded cDNA. Transposase activity was halted using QG buffer (Qiagen), and fragmented cDNA was purified on AMPure XP beads. Indexed sequencing libraries were PCR-amplified and sequenced for 50-bp paired-end reads on an Illumina HiSeq 2000 instrument at the Duke Genome Sequencing Shared Resource and for 75 paired-end reads on an Illumina MiSeq. Reads were trimmed to 50 bp, and sequences that aligned to the delivered lentiviral vector were removed from analysis using Bowtie2 (ref. 47). Filtered reads were then aligned to human RefSeq transcripts using Bowtie2. Statistical analysis, including multiple-hypothesis testing, on three independent biological replicates was performed using DESeq48.
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Chromatin immunoprecipitation. ChIP experiments were per-formed in in biological triplicate, starting from independent cell transductions, and harvested 7 d after transduction. For each rep-licate, 2 × 107 nuclei were resuspended in 1 mL of RIPA buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS in PBS at pH 7.4). Samples were sonicated using a Diagenode Bioruptor XL sonicator at 4 °C to fragment chromatin to 200–500-bp segments. Insoluble components were removed by centrifugation for 15 min at 15,000 r.p.m. We conjugated 5 µg of anti-FOSL1 (Santa Cruz Biotechnology, sc-183), anti–GATA-2 (Santa Cruz Biotechnology, sc-9008), anti-Flag (Sigma-Aldrich, F1804), or anti-H3K9me3 (Abcam, ab8898) to 200 µl of either sheep anti-rabbit or sheep anti-mouse IgG magnetic beads (Life Technologies, 11203D/11201D). Sheared chromatin in RIPA buffer was then added to the antibody-conjugated beads and incubated on a rotator overnight at 4 °C. After incubation, beads were washed five times with an LiCl wash buffer (100 mM Tris, pH 7.5, 500 mM LiCl, 1% NP-40, 1% sodium deoxycholate), and remaining ions were removed with a single wash with 1 mL of TE (10 mM Tris-HCl, pH 7.5, 0.1 mM Na2-EDTA) at 4 °C. Chromatin and antibodies were eluted from beads by incubation for 1 h at 65 °C in immunoprecipitation elution buffer (1% SDS, 0.1 M NaHCO3) followed by overnight incu-bation at 65 °C to reverse formaldehyde cross-links. DNA was purified using MinElute DNA purification columns (Qiagen).
ChIP-qPCR. qRT-PCR using QuantIT Perfecta Supermix was per-formed with the CFX96 Real-Time PCR Detection System (Bio-Rad) and the oligonucleotide primers reported in Supplementary Table 16. 100 pg of ChIP DNA was loaded into each reaction. The results are expressed as a fold-increase of signal at the HS2 enhancer normalized to signal at the GAPDH promoter by the ∆∆Ct method.
ChIP sequencing. Illumina TruSeq adapted libraries were con-structed using an Apollo 324 NGS Library Prep System with a PrepX Complete ILMN DNA Library Kit (WaferGen Biosystems). ChIP products were amplified with 15 cycles of PCR, and frag-ments 150–700 bp in length were selected using an AxyPrep MAG PCR Clean-Up Kit (Axygen MAG-PCR-CL-50). Libraries were sequenced using single end 50-bp reads on an Illumina HiSeq at the Duke Genome Sequencing Shared Resource. ChIP-seq analyses were executed independently for each epitope to find regions of differential Flag or H3K9me3 enrichment compared with cells treated with lentivirus encoding dCas9-KRAB without sgRNA or dCas9 with sgRNA. To account for background levels of inte-grated lentiviral DNA, we filtered reads aligning to the delivered lentiviral sequences from analyses using Bowtie2 (ref. 47). Next we used Bowtie2 to align the remaining reads to the hg19
reference genome and removed PCR duplicates using the rmdup tool from SAMtools49. Reads from each triplicate for each con-dition were combined, and peaks were called using MACS50. Resulting peaks from each condition with a q-value ≤ 0.05 were merged using the mergeBed tool from BEDTools51. The number of reads overlapping each of these peaks for each triplicate was deter-mined using the intersectBed tool from BEDTools. Differential enrichment for each peak was evaluated from these read counts using DESeq2 (ref. 52) with an FDR cutoff of ≤0.05.
DNase I–hypersensitivity sequencing. All experiments were per-formed in biological triplicate, starting from independent cell transductions. Library preparation and analysis were performed as previously described with the one exception of addition of a 5′ phosphate group to oligo 1b to increase ligation efficiency53. For each replicate, 7 d after transduction, approximately 2.5 × 107 nuclei were extracted and then digested with different amounts of DNase I for 16 min at 37 °C. Reactions were ter-minated by the addition of 50 mM EDTA. Libraries were con-structed from pooled digests as described and sequenced on the Illumina HiSeq2000 platform with 50-bp single-end reads at the Duke Genome Sequencing Shared Resource. Resulting reads were filtered for delivered vector sequences and aligned by Bowtie54. Peaks were called by MACS version 2 (ref. 50), with a cutoff line at FDR < 0.01. Differential DHS sites were determined using DESeq2 (ref. 52).
44. Kabadi, A.M., Ousterout, D.G., Hilton, I.B. & Gersbach, C.A. Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector. Nucleic Acids Res. 42, e147 (2014).
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